Greener Approach To Prepare Electrospun Antibacterial Î²

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A greener approach to prepare electrospun antibacterial #-cyclodextrin/ cellulose acetate nanofibres for removal of bacteria from water Lebea Nathnael Nthunya, Monaheng L. Masheane, Soraya P. Malinga, Edward N. Nxumalo, Tobias G. Barnard, Mahalieo Kao, Zikhona N. Tetana, and Sabelo D. Mhlanga ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b01089 • Publication Date (Web): 05 Dec 2016 Downloaded from http://pubs.acs.org on December 5, 2016

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ACS Sustainable Chemistry & Engineering

A greener approach to prepare electrospun antibacterial β-cyclodextrin/ cellulose acetate nanofibres for removal of bacteria from water Lebea N. Nthunyaa,b, Monaheng L. Masheanea,b, Soraya P. Malingaa, Edward N. Nxumalob, Tobias G. Barnardc, Mahalieo Kaod, Zikhona N. Tetanad,e, Sabelo D. Mhlangab* a

Department of Applied Chemistry and the DST/Mintek Nanotechnology Innovation Centre-

Water Research Node, University of Johannesburg, P.O. Box 17011, Doornfontein, 2028, Johannesburg, South Africa b

Nanotechnology and Water Sustainability Research Unit, College of Science, Engineering and

Technology, University of South Africa, Florida, 1709, Johannesburg, South Africa c

Water and Health Research Centre, University of Johannesburg, P.O. Box 17011,

Doornfontein, 2028, Johannesburg, South Africa d

Molecular Sciences Institute, School of Chemistry, University of the Witwatersrand, P.O. Wits,

Johannesburg, 2050, South Africa e

Microscopy and Microanalysis Unit, University of the Witwatersrand, P.O. Wits, Johannesburg,

2050, South Africa Corresponding author: Email: [email protected]; Tel: (+2711) 471 2104

ACS Paragon Plus Environment

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Abstract This work reports the synthesis of β-cyclodextrin (β-CD)/cellulose (CA) nanofibres embedded with silver (Ag) and silver/iron (Ag/Fe) nanoparticles (NPs) via a benign process involving insitu electrospinning of the biopolymers with Ag and Fe salts. The electrospun nanocomposite fibres containing Ag+/Fe3+ ions were then subjected to UV photochemical reduction in the presence of water vapour under inert atmosphere to reduce the ions to zero-valent state. SEM and TEM revealed that the average diameter of the β-CD/CA nanofibres was 382.12 ± 30.09 nm and the diameters of Ag and Ag/Fe NPs were 38.81 ± 8.21 nm and 56.29 ± 12.64 nm respectively after reduction. The XRD and EDS analysis confirmed the presence of the Ag and Fe NPs on the surface of the nanofibres. The effect of UV irradiation time on the reduction of the Ag+ and Fe3+ was studied by measuring the UV-Vis absorbance of the reduced NPs. The biocidal effect of Ag and Ag/Fe was investigated using twelve different strains of bacteria. The Ag and Ag/Fe NPs embedded on the β-CD/CA nanofibres exhibited strong biocidal effect on all the bacteria strains.

Key words: β-cyclodextrins, bacteria, cellulose acetate, nanofibres, silver/iron nanoparticles, water purification


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INTRODUCTION The rate of mortalities occurring as a result of bacterial infections is a daunting statistic, especially in low income/rural communities where there is no provision of safe drinking water or adequate sanitation services. The estimated worldwide deaths of more than 10 million people per year in developing countries are related to bacterial infections.1 Various types of Gram-positive and Gram-negative bacteria are distributed in the environment and they are the cause of numerous diseases including diarrhea,2,3 vomiting,4 and fatal hemolytic uremic.4 There is a need to develop appropriate technologies and the improvement of various existing remedial technologies in order to suit application in specific areas with unique problems. Nanomaterials such as silver (Ag) and iron (Fe) nanoparticles (NPs) have been developed to prevent bacterial infections that emerge from polluted water.5 However, there are problems that relate to the toxicity of nanomaterials and their leaching to the environment that need to be overcome. The way to go is to prevent diseases associated with pathogens using sustainable means (i.e. sustainable processes and materials) without creating further problems.6,7 The approach is to develop solutions prepared from environmentally benign materials using green synthesis strategies, i.e. by avoiding the use toxic chemicals while maintaining the efficiency and effectiveness of the materials produced.8 Nanomaterials are of great interest in recent technologies because they exhibit superior/desirable properties that are different from those of their macroscopic counterparts.3,9,10 Many researches have shown that the size of NPs and their contact with bacteria play a critical role in their bactericidal effects.11,12 As such, different NPs, with no exception to Ag NPs, have received considerable attention due to their remarkable antibacterial properties.13 NPs such as Ag and Fe supported on different materials including nanofibrous biopolymers such as cyclodextrins


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(CDs) and cellulose acetate (CA) have been used in a wide range of applications including wound dressing,14 coating of the surgical apparatus,15 textiles,16 cosmetics,17 food packaging18 and water purification.19,20 The choice of the materials with no exception to CD and CA supporting the NPs depends on a number of factors which include toxicity, availability, biocompatibility, biodegradability and cost. 21,22 Electrospun nanofibrous polymers as supporting materials for NPs have found intense application in water treatment 23. This is due to their high surface area to volume ratio, pore sizes at the nanoscale, easy control of porosity and easy modification of their surface chemistry.24,25 The NPs supported on nanofibres can be prepared using different strategies including in-situ polymerization, where the metal ions are added to the polymer and reduced to NPs from the electrospun nanofibres and ex-situ polymerization where the prepared NPs are added to the polymer prior to electrospinning. It is believed that the distribution of NPs on the surface of the supporting polymer affects the biocidal functionality of the nanofibrous composite.26 The cost of the Ag NPs can be reduced by the addition of magnetic NPs such as Fe NPs while maintaining bacterial toxicity of the biocides since Fe sources are less expensive compared to Ag sources. The inherent magnetic behaviour of Fe helps in the removal of other NPs, in particular, those found to leach out of the supporting polymers in water bodies.27 Photoreduction12 and photochemical reduction28 have been used for the synthesis of Ag NPs using UV irradiation. However, to the best of our knowledge, no work has been reported on the UV reduction of Ag and Ag/Fe supported on electrospun β-CD/CA nanofibers in the presence ionized water vapour. In this work, the UV/ionized water vapour reduction of Ag and Ag/Fe NPs supported on the β-CD/CA nanofibres is demonstrated. The nanofibres were further investigated for their antibacterial activity using B. cereus and E. coli strains to determine the effect of contact


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of both Gram negative and Gram-positive bacteria with the NPs. Furthermore, twelve different strains of bacteria were used determine the minimum inhibition concentration (MIC) of the antibacterial nanofibres.

EXPERIMENTAL Materials β-CDs (Mw = 1134.98 g/mol), CA (39.8 wt% acetyl, Mw = 30, 000 g/mol), Iron(III) chloride hexahydrate (FeCl3•6H2O) (ACS reagent grade), Ethylenediaminetetraacetic acid (EDTA – ACS reagent grade) acetone (HPLC grade), N,N-dimethyl acetamide (DMAc), Mueller Hinton broth (for microbiology), agar (for microbiology), p-iodonitrotetrazolium chloride (BioReagent grade) were purchased from Sigma Aldrich (South Africa). B. cereus, E. faecalis, E. coli, K. pneumonia, K. oxytoca, P. aeruginosa, P. mirabilis, S. boydii, S. sonnei, and E. cloacae American Type Culture Collection (ATCC) strains were obtained from National Health Laboratory Services (NHLS) in South Africa. Silver nitrate (AgNO3) (reagentPlus® grade) was purchased from Rochelle Chemicals, South Africa. All chemicals and reagents were used as received.

Electrospinning of nanofibres The synthesis of Ag and Ag/Fe NPs embedded on the β-CD/CA (1:1) nanofibres was conducted using in-situ polymerization during electrospinning. β-CD/CA (1:1) was found to be the optimum polymer ratio needed to produce antibacterial nanofibres that were non-beaded. AgNO3 and β-CD/CA were dissolved in a mixed solvent system of acetone and DMAc (3:2


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solvent ratio) for dispersion of Ag on β-CD/CA nanofibres. AgNO3, FeCl3 and β-CD/CA were dissolved in the similar mixed solvent system of acetone and DMAc (3:2) for dispersion of Ag/Fe NPs on β-CD/CA nanofibres. The amount of metal ion salts was calculated to give 4wt% of Ag NPs and 2wt% of individual Ag/Fe NPs (that is 2wt% Ag NPs and 2wt% Fe NPs) relative to the β-CD/CA polymer powders. The concentration of polymers used was 32% (m/v) at the ratio of 1:1 of β-CD:CA. The prepared solutions were transferred to a 10 mL syringe fitted with needle of 0.8 mm internal diameter. The syringe was placed on a NE-4000 double syringe pump. A high voltage generator (EV11M, TEL Atomic) was used to induce an electric field between the collecting plate and the tip of the needle. The nanofibres were synthesized at the following electrospinning conditions: flow rate of 0.7 mL/h, a distance of 15 cm between the aluminium collecting plate and the tip of the needle, and voltage of 16 kV at room temperature.

UV-assisted reduction of the Ag+ and Fe3+ ions on β-CD/CA nanofibres The electrospun β-CD/CA nanofibres embedded with Ag+ and Ag+/Fe3+ ions were irradiated with UV light at λ max = 249 nm using a 420 W Hg lamp (model no. 3040, Photochemical reactors Ltd) fitted in a horizontal furnace (Figure 1). The reduction of the metal ions into NPs was carried out at 70 ºC in the presence of water vapour to enhance the photochemical reduction of the metal ions to NPs. The reduction can also be achieved in the presence of small amounts of ammonia but this is not preferred. The nanofibres were irradiated at different times from 30 min to 210 min in order to determine the effect of the irradiation time on the intensity of NPs dispersed on the surface of nanofibres. The antibacterial nanofibres were finally cross-linked with EDTA following a method reported by Zhao and the co-workers to enhance their usability in water treatment.29


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Fan system

UV lamp

Pyrex tube with opening above sample

UV rays path to sample Furnace Gas inlet

Nanofibre mats (sample)

UV rays striking water vapour and trigger release of electrons

Thermocouple

Figure 1. The experimental set-up for the UV-assisted reduction of Ag and Ag/Fe NPs supported on β-CD/CA nanofibres.

Characterization and testing of the β-CD/CA antimicrobial nanofibres The surface morphology of the nanofibres was studied using a scanning electron microscope (TESCAN-x max 20mm2). The samples were cut into 1 mm x 1 mm dimensional sizes and coated with carbon under vacuum using a carbon coater (Quadrum Q150TE). The SEM images were acquired at the operating voltage of 20 kV. X-ray diffraction (XRD) patterns of the nanofibres were acquired with a Rigaku Ultimate IV diffractometer equipped with a Cu Kα radiation source, a scintillation counter detector and a K-β filter. The XRD patterns were analyzed qualitatively using “PDXL” software, provided with a JCPDS-PDF2 database. The


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scanning was run in the 2θ° = 0 – 90° at the speed of 0.5°·min-1. To get optimized peak-tobackground ratios, the current and voltage were set at 30 mA and 40 kV respectively. Thereafter, 2 g of crushed nanofibres was used for XRD analyses. UV-Vis spectroscopy (Perkin Elmer Shimadzu 2450 spectrophotometer) was used to determine the UV absorption of Ag and Ag/Fe NPs deposited on the β-CD/CA nanofibres and to determine their quantity (abundance) with respect to change in UV irradiation time. This was achieved by crushing 1 g of nanofibres and thoroughly mixed with 1 g of BaSO4. The sample and the blank (BaSO4) were transferred into the chamber and the samples were analyzed at the absorbance mode, 5 nm slit width and a medium scan rate at a wavelength of 200 – 900 nm. Transmission electron microscopy (TEM) studies for the dispersion of the Ag and Ag/Fe NPs on the β-CD/CA nanofibres were performed at an acceleration voltage of 200 kV by using a Jeol JEM-2100F Field Emission Electron Microscope equipped with a LaB6 source, energy dispersive X-ray spectroscopy (EDS) detector and CCD cameras. The TEM samples were prepared by depositing small amount of synthesized nanofibres onto a TEM grid (200 mesh size Cu-grid) coated with a lacy carbon film. The elemental composition of the samples was analyzed by X-ray energy dispersive spectroscopy (EDS) detector at an operating voltage of 15 kV. The antimicrobial activity of the β-CD/CA nanofibres embedded with Ag and Ag/Fe NPs was tested against Gram positive B. cereus, Gram positive E. faecalis, Gram negative E. coli, Gram negative K. pneumonia, Gram negative K. oxytoca, Gram negative P. aeruginosa, Gram negative P. mirabilis, Gram negative S. boydii, Gram negative S. sonnei, and Gram negative E. cloacae. The bacterial strains were grown on a plate and maintained on a Mueller-Hinton agar during experiments. The plates were incubated at 37 ºC for 24 h. The strains were grown in a liquid culture by inoculating Mueller-Hinton broth with the bacterial colony of interest. All strains were


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grown at 37 ºC with constant mild shaking until an optical density (OD600) of 0.6 at 600 nm was reached. The basic disc diffusion test was used to test the antibacterial activity of β-CD/CA nanofibres embedded with Ag and Ag/Fe NPs. A bacterial suspension (100 µL) with OD600 of 0.6 was evenly spread on plastic petri dishes with an agar of thickness of approximately 4 mm. The antibacterial nanofibres that were cut to similar dimensions were placed onto the plates to have contact with bacteria. The plates with the nanofibres were incubated at 37 ºC for 16 h. The nanofibres were removed and the area of bacterial growth inhibition following contact with nanofibre mats was assessed. The minimum inhibitory concentration (MIC) tests were done in a sterile 96 well micro titre plates with lids. Test samples used to determine the minimum inhibitory concentration (MIC) were prepared by evenly dispersing 0.5 g of the antibacterial β-CD/CA nanofibres in 20 mL of water. Then 100 µL of this test solution was added to the first well and serial diluted by transferring 50 µL of the dilution to the next well containing 50 µL media. Thereafter, 50 µL of bacterial cultures with similar OD600 was added to each well containing the test samples. The micro titre plates were closed and incubated at 37 ºC for 24 h. To indicate the bacterial growth, 50 µL of p-iodonitrotetrazolium chloride was added to each well and incubated for 40 min using Eloff JN method.30 The wells that turned purple indicated the presence of bacteria, hence no growth inhibition. All appropriate positive, negative and reagent controls were included to ensure the reliability of the test.


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RESULTS AND DISCUSSION SEM analysis The SEM micrographs of the β-CD/CA nanofibres obtained at the optimum electrospinning conditions are shown in Figure 2. It was observed that at polymer concentrations below and/or above 32% (m/v) and blending ratios different from 1:1 (β-CD/CA), the nanofibres were formed concurrently with beads and electrospraying.

This phenomenon was associated to critical

polymer concentration/viscosity that failed to sustain the stretching effect of polymer jet.31 This suggested that the minimum concentration/viscosity of the polymers at the optimum ratios was a key solution to the formation of free-beaded nanofibres. Thus a β-CD:CA blend (32% (m/v)) at the ratio of 1:1 was the characteristic polymer concentration and the ratio that assisted in the stabilization of the bending of the jet in the electric field, thus allowing the molecular entanglement for the formation of free-beaded nanofibres. Other parameters such as the solution injection flow rate, the distance between the tip of the spinneret and the collector and the voltage were also taken into consideration to determine their effect on the morphology of the nanofibres. At flow rates lower than 0.5 mL.h-1, the polymer jet was found to cut several times due to the insufficient polymer solution injected out of the tip of the spinneret to be stretched by the electric field. As the flow rate was increased beyond 0.5 mL.h-1, the diameter of the nanofibres increased significantly. As such, 0.5 mL.h-1 was chosen as the optimum flow rate that produced uniform nanofibres (430 nm in diameter). The uniformity of the nanofibres was also maintained at 15 cm as the distance between the tip of the spinneret and the collector and the voltage of 14 kV. This was related to the characteristic electric field (voltage per distance)32 that was suffient enough to produce free-beaded β-CD:CA (32 %(m/v)). The average diameter of free-beaded nanofibres obtained at the 0.5 mL.h-1, 15 cm and 14 kV as


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the optimum electrospinning parameters was found to be 382.12 ± 30.09 nm which can comfortably be reported as the average diameter of the nanofibres used in this study. The pores observed from the intertwined nanofibre mats obtained at optimum conditions (Figure 2) is an indication of the highly porous nature of the nanofibers which can act as nanofilters that can be used for water filtration at low operational pressures.

Figure 2. SEM images of β-CD/CA nanofibres obtained at optimum electrospinning conditions: (a) flow rate = 0.5 mL.h-1, (b) distance = 12 cm and (c) voltage = 14 kV.

Photochemical reduction of Ag+ ions and Fe3+ ions in the β-CD/CA nanofibres To achieve good dispersion while maintaining strong interaction between the NPs and nanofibres, the active Ag or Ag/Fe NPs were fabricated by reducing the metal ions on the nanofibres using UV irradiation under induced water vapour in the presence of N2 gas after electrospinning. When the nanofibres were irradiated with UV light at 249 nm under a constant flow of N2 gas, the colour of the Ag-β-CD/CA nanofibres changed from white to dark brown while the colour of the Ag/Fe-β-CD/CA nanofibres changed from white to yellow. The colour changes gave an indication that the Ag+ and Fe3+ ions on the β-CD/CA nanofibres were reduced


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to Ag0 and Fe0 respectively. During UV irradiation, water vapour ionizes to form the H2O+ and releases the electrons which are accepted by the positively charged Ag+ and Fe3+ ions, hence reduce the metal ions to their respective NPs.33 When metal ions receive the electrons, they are reduced to Ag0 and Fe0 as shown in Equations 1 and 2. The reduction of the metals ions was confirmed by XRD, UV-Vis, TEM and EDS. The results are discussed in the next sections. H2O + hv → H2O+ + eAg+ + e- → Ag0

(1)

and Fe3+ + 3e- → Fe0

(2)

XRD analysis Figure 3 shows the XRD patterns of Ag and Ag/Fe NPs embedded on β-CD/CA nanofibres. The JCPDS card values for the following planes (111), (200), (220), (311) and (222) at 2 θ = 38.28º, 44.49º, 64.75º, 77.79º and 81.92º on β-CD/CA nanofibres are characteristic diffractions of Ag NPs (Figure 3(a)). The diffraction patterns of Ag NPs shifted to 2 θ = 38.19º, 44.39º, 64.58º, 77.57º and 81.73° on the same JCPDS values upon addition of Fe (Figure 3(b)). This was expected since the chemical surrounding of Ag NPs was affected by the addition of the Fe NPs, which in turn changed their shape, size and electron density.34 The shift was also attributed to extended defects and the micro strains of the peak shapes and widths of the NPs. NPs with diameters less than 100 nm are known to have defects on their surface.35 The diffractions at 2 θ = 20.44º on Figure 3b and 2 θ = 21.84º on Figure 3a were associated to the carbon allotropes in the nanofibres.


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Figure 3. XRD patterns of Ag and Ag/Fe NPs on β-CD/CA nanofibres: (a) Ag NPs and (b) Ag/Fe NPs. UV-Vis analysis The UV-Vis absorption spectra of Ag and Ag/Fe NPs on the β-CD/CA nanofibres are shown in Figure 4. The maximum absorbance peaks of Ag NPs occured at the wavelength range of 399 nm – 426 nm, which corresponds to the surface plasmon band of Ag NPs. The surface plasmon resonance increased as the holding time of UV irradiation was increased (Figure 4a). The increase in surface resonance indicated that the density of the NPs was produced increasing with increase in irradiation time. However, the rate of Ag+ and Fe3+ ions reduction became less rapid at higher reduction times (between 120 min and 210 min) (Figure 4a,b). This was due to depletion of metal ions that were rapidly reduced at the start of the UV irradiation process.


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Figure 4. UV-Vis absorbance spectra of NPs supported on electrospun β-CD/CA nanofibres reduced at different irradiation times. Inserted graphs are the maximum absorbance of NPs with respect to UV irradiation time. (a) Ag NPs and (b) Ag/Fe NPs.


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The absorption peaks broadened as the UV irradiation time was increased. This suggested that the size distribution of the NPs changed as the time of UV irradiation was increased (i.e. they became bigger). The UV-Vis absorbance peaks of Ag/Fe NPs were observed at the maximum wavelength of 326 nm at lower reduction times. The maximum absorbance peaks shifted from lower wavelengths to higher wavelengths (326 nm – 361 nm) as the UV irradiation time was increased from 30 min to 210 min (Figure 4b). This wavelength shift however did not affect the narrow and symmetric absorbance peaks which implied the non-changing distribution of the NPs on the hosting material (nanofibres). The surface plasmon resonance absorption of Ag/Fe NPs also increased with an increase in irradiation time, as was observed with Ag NPs. This was due to the increase in density of the reduced Ag/Fe NPs with time. TEM and EDS analysis The presence of elemental Ag and Fe on the nanofibres was also confirmed by EDS analysis (Ag at 3.0 kV and Fe at 6.2 kV) Figure 5(i) and 6(i). The intense signals observed for C and O were due to C and O atoms present in the β-CD/CA nanofibres while Cr and Cu were components of the TEM instrument (Cr forms part of the Cr alloys in the instrument while Cu comes from the Cu grid that was used as a sample holder. The intense peaks of Cr and Cu overshadow the peaks of Ag and Fe NPs since Ag and Fe ions were added in small quantities (4wt%) during the synthesis of the antimicrobial β-CD/CA nanofibres. Figure 5(ii) and 6(ii) present TEM images of the Ag and Ag/Fe NPs on the β-CD/CA nanofibres with change in UV irradiation time. The density of Ag and Ag/Fe NPs increased with an increase in UV irradiation time. The size distribution of Ag NPs was narrower than that of Ag/Fe NPs as the UV irradiation time was increased. Figure 7 shows the effect of UV irradiation


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time on the average size of NPs. The average sizes of Ag NPs were found to be 30 ± 7 nm and 52 ± 5 nm at 30 min and 210 min respectively. The average sizes of Ag/Fe NPs were found to increase from 30 min to 90 min (44 ± 4 nm – 72 ± 7 nm). The size ranges of the synthesized Ag and Ag/Fe NPs supported on the β-CD/CA nanofibres exhibit the possibility of sizes needed to produce significant biotoxicity to pathogens.

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Figure 5. (i) TEM images of the Ag NPs on β-CD/CA nanofibres reduced at different UV irradiation times: (a) 30 min, (b) 60 min, (c) 90 min, (d) 120 min and (e) 210 min and (ii) the corresponding EDS.

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Figure 6. (i) TEM images of the Ag/Fe NPs on β-CD/CA nanofibres reduced at different UV irradiation times: (a) 30 min, (b) 60 min, (c) 90 min, (d) 120 min and (e) 210 min and (ii) the corresponding EDS.

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Figure 7. The effect of UV irradiation time on the size of Ag and Ag/Fe NPs on β-CD/CA nanofibres.


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Antibacterial tests of the Ag and Ag/Fe on β-CD/CA nanofibres The antibacterial Ag and Ag/Fe NPs supported on β-CD/CA nanofibres were tested against representatives of Gram-positive (B. cereus) and Gram-negative (E. coli.) bacteria. Figure 8 shows the biocidal effect of β-CD/CA nanofibres containing Ag and Ag/Fe NPs against Grampositive B. cereus and Gram-negative E. coli using the disc diffusion plate method. The growth inhibition of both B. cereus and E. coli can be clearly observed on Figure 8(a,b). The nanofibre mat C that was used as a control did not show an inhibitory effect of the growth on the bacterial strains, which implied that the Ag and Ag/Fe NPs embedded on the β-CD/CA nanofibres are the fundamental antibacterial agents that induce the bacterial destruction. The small inhibition zone observed on nanofibre mat 1 and 3 on Figure 8(a,b) was associated with the presence of Ag NPs on the surface of the nanofibres. However, all the nanofibre mats did not show a direct measurable zone of bacterial inhibition since the nanofibres bound antibacterial agents could not diffuse into agar as per normal test. This was associated to fixed attachment and too low concentrations of the NPs on the nanofibres. Therefore, there was no diffusion of the NPs that produce a measurable inhibition zone, which implied that the antibacterial NPs did not leach out of the nanofibres as required in water treatment.


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Figure 8. Spread plate method showing growth inhibition of different bacterial strains under Ag and Ag/Fe NPs on β-CD/CA nanofibres where 1 and 2 are C nanofibre mats decorated with Ag NPs while 3 and 4 are nanofibre mats decorated with Ag/Fe and C is the nanofibres without NPs (control): (a) Gram-positive B. cereus and (b) Gram-negative E. coli.

Since there was no concrete evidence that could be associated with the antibacterial activity of the β-CD/CA nanofibres containing Ag and Ag/Fe NPs and the normal concentrations of the NPs needed to inhibit bacterial growth, it was decided to test the antibacterial potential of the Ag and Ag/Fe using a 96 well plate assay. It was observed that the bacterial growth inhibition could be accurately measured using a serial dilution of the ground β-CD/CA nanofibre mats that contained Ag and Ag/Fe NPs. The MIC that produced observable antibacterial activity was also tested against B. cereus, E. faecalis, E. coli, K. pneumonia, K. oxytoca, P. aeruginosa, P. mirabilis, S. boydii, S. sonnei, and E. cloacae strains as shown on Figure 9. Two E. coli strains and two K. pneumonia strains with different ATCC numbers were used in this test. MIC provides information about the resistance of bacterial strains against antibacterial agents.36 The β-CD/CA nanofibres (6.25 mg.mL-1) that contained 0.25 mg.mL-1 of Ag exhibited inhibition of the growth


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on all strains of bacteria as evidenced by the absence of the purple colour. The β-CD/CA nanofibres (3.125 mg.mL-1) that contained 0.125 mg/mL of Ag further exhibited inhibition of the growth on E. faecalis, P. mirabilis strains. The β-CD/CA nanofibres containing Ag/Fe as bacterial agents exhibited growth inhibition to all bacterial strains at the similar concentrations of the nanofibres containing Ag NPs. Since the Ag/Fe NPs were embedded on the nanofibres at 2wt% Ag and 2wt% Fe in order to maintain the 4wt% of NPs, it transpired that Fe NPs assisted in the antibacterial activity of Ag NPs; since lower concentrations of Ag with Fe were used compared to the concentration of Ag without Fe.

The minimum concentration of the antibacterial agents that showed effective antibacterial activity on E. faecalis and P. mirabilis was found to be lower than the concentration that showed antibacterial activity on B. cereus, E. coli, K. pneumonia, K. oxytoca, P. aeruginosa, S. boydii, and S. sonnei (Figure 9) i.e., the concentration of antibacterial agents needed to produce a noticeable antibacterial activity on B. cereus, E. coli, K. pneumonia, K. oxytoca, P. aeruginosa, S. boydii, and S. sonnei was higher than the concentration needed to inhibit the growth of E. faecalis and P. mirabilis. The differences in the required concentration of the antibacterial agents needed to produce noticeable antibacterial activity was associated with the response and resistance of these strains linked to their bacterial structure, bacterial cell wall, cell membrane, and thickness of their peptidoglyacan layer, which respond differently to different bacteria and thus help them resist antibacterial agents differently. Despite this, the MIC could be reported as the nanofibres (6.25 mg/mL) that contained 0.25 mg/mL of Ag and Ag/Fe NPs.


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a

b

Figure 9. MIC of antibacterial β-CD/CA nanofibres embedded with (a) Ag NPs and (b) Ag/Fe NPs on 12 strains of bacteria.

CONCLUSION In conclusion, in-situ electrospun Ag and Ag/Fe NPs embedded on β-CD/CA nanofibres were successfully synthesized using electropinning and activated using greener UV photochemical reduction process in the presence N2 gas under ionized water vapour. This process of is comparatively safer since ˃ 90% of the reagents used are environmentally friendly and does not involve multiple steps of intensive chemicals. During the use of UV rays to reduce the Ag and


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Ag/Fe NPs the use of toxic reducing reagents like sodium borohydride is avoided. The number of visible (under TEM) NPs was found to increase with an increase in UV irradiation time. The Ag and Ag/Fe NPs supported on β-CD/CA nanofibres exhibited similar biocidal effects on all bacterial strains used in this study. The good antibacterial activity of the Ag/Fe NPs on the βCD/CA nanofibres was also found to be cost effective since Ag salts are much more expensive than the Fe salts; the addition of Fe NPs reduced the amount of Ag used while maintaining the antibacterial effect of the NPs, thus keeping the synthesized antibacterial agents. Generally, the NPs exhibited good bactericidal properties on all strains of bacteria at different MICs which make them ideal for antibacterial purification of water. In summary, the study has shown that the reduction of in-situ electrospun Ag-β-CD/CA and Ag/Fe-β-CD/CA nanofibers can be reduced under ambient conditions of UV-irradiation in the presence of water vapour. The resulting nanofibrous composite did not show leakage of the nanoparticles to the environment in the performed tests, but needs further confirmation when used for water treatment. The process is greener and this has a bearing on the sustainability of the water treatment solutions derived from the materials.

ACKNOWLEDGEMENTS The authors are grateful to the DST/NRF Nanotechnology Flagship Project (No. 97823), the Water Research Commission, the DST/Mintek Nanotechnology Innovation Centre (NIC), Department of Applied Chemistry (University of Johannesburg) and the NanoWS Research Unit, College of Science, Engineering and Technology (NanoWS4RUMP CE Project, University of South Africa) for supporting this work.


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For Table of Contents Use Only

A greener approach to prepare electrospun antibacterial β-cyclodextrin/ cellulose acetate nanofibres for removal of bacteria from water Lebea N. Nthunya, Monaheng L. Masheane, Soraya P. Malinga, Edward N. Nxumalo, Tobias G. Barnard, Mahalieo Kao, Zikhona N. Tetana, Sabelo D. Mhlanga*

Synopsis A sustainable procedure for the synthesis of environmentally benign antibacterial nanofibres using in-situ electrospinning followed by UV-reduction assisted with ionized water vapour is reported.


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Greener Approach To Prepare Electrospun Antibacterial Î²

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